The present invention relates to a device for the detection of a fluorescent dye in a sample, comprising a radiation source means with the aid of which light for exciting the fluorescent dye to be detected can be transmitted into the sample applied to a carrier, and a detecting means for detecting fluorescent light which has been emitted by the fluorescent dye to be detected.
Such devices in which fluorescent light is detected by a photomultiplier means are used e.g. for analyses in the field of molecular biology and genetic engineering. For this purpose, a sample is applied to a carrier and temporarily brought into contact with a fluorescent marker. Those substances to be analyzed which have an affinity for the marker agglutinate the marker and, consequently, they can be excited to emit fluorescent light. It follows that, due to the excitability of fluorescence, the marker-agglutinating property of the substance to be analyzed becomes visible, whereby conclusions can be drawn with regard to the nature of the sample material.
However, a disadvantage of the devices known from the prior art is to be seen in the fact that the fluorescent light, which has no preferred direction and which is therefore emitted into the whole solid angle, can only be detected from a small solid angle area with the aid of a photomultiplier means. It follows that the detection sensitivity of the known devices is only very small.
In view of this disadvantage of the devices according to the prior art, it is the object of the present invention to improve the known device for the detection of a fluorescent dye in such a way that its detection sensitivity is increased.
This object is achieved by a device of the type cited at the start, which is characterized by a hollow space having an internal high-reflectance surface, a first aperture directed towards the sample, and a second aperture located opposite the detecting means.
Due to the fact that the means for exciting the fluorescent dye to be detected, the surface of which has the sample applied thereto, is located opposite the first aperture of the hollow space, the fluorescent light is emitted from a large solid angle into the hollow space. Since the hollow space is additionally provided with an internal high-reflectance surface, the light transmitted into the hollow space can propagate in said hollow space until it is detected by a detector.
It follows that, in comparison with the prior art, the fluorescent light can be detected from a much larger solid angle, whereby the detection sensitivity of the device is greatly increased in comparison with the known devices.
According to a preferred embodiment of the present invention, the high-reflectance surface consists of barium sulfate or spectralon. Such surfaces have a reflectance of up to 99.8%. Hence, the reflectance losses in the hollow space can be kept very small.
According to a preferred embodiment, the radiation source means can be provided in such a way that it emits monochromatic light, especially laser light. The advantage of monochromatic light is to be seen in the fact that the influence of monochromatic light on the fluorescent light emitted can be controlled much better, e.g. by purposeful absorption. The advantage of laser light is the comparatively high spot intensity which can be achieved by means of the laser light. Correspondingly high excitation rates of the fluorescent dye to be detected are therefore obtained.
Furthermore, a beam forming means, especially a beam expansion means, can be provided after the radiation source means. This measure permits an adaptation of the light beam emitted by the radiation source means to the dimensions of the desired laser spot size.
According to a preferred embodiment, the radiation source means and the carrier can have provided between them a scanning means with the aid of which rays emitted by the radiation source can be guided over the carrier. A plurality of samples applied to the carrier in the form of an array can, in this way, sequentially be excited to exhibit fluorescence and the resultant fluorescent light can be detected without any necessity of carrying out a time-consuming exchange of samples, including the cleaning of the carrier and the like, or a mechanical displacement of the sample relative to the carrier.
According to another advantageous embodiment, a focussing lens system is provided for focussing the excitation light onto the sample. The spot intensity of the excitation light can be increased still further in this way, whereby the resolution and intensity of the secondary light is increased.
According to an advantageous embodiment, the focussing lens system is an F/xcex8 lens in the case of which an image of the scanning ray bundle is formed according to the so-called F/xcex8 condition yxe2x80x2=Fxxcex8, wherein yxe2x80x2 is the imaging coordinate, F the focal length and xcex8 the angle included by the scanning ray bundle and the optical axis. This permits a focussing of beams of light independently of the distance to the optical axis, and the focussing effect can therefore be intensified in comparison with conventional lenses. A change of the angle of the deflection mirror is therefore converted into a proportional deviation yxe2x80x2.
According to another advantageous further development of all the above-mentioned embodiments, a beam divider can be provided in such a way that, by means of said beam divider, part of the light emitted by the radiation source means can be directed onto the sample and part of the fluorescent light emitted by the fluorescent dye to be detected can be conducted into the hollow space. By means of this measure, a compact and easily operable setup of the device according to the present invention can be realized.
In this connection, the beam divider can be provided in the form of a dichroic beam divider which, on the one hand, reflects the excitation light with high efficiency and deflects therefore most of the excitation light towards the sample and, on the other hand, permits passage of the fluorescent light with high efficiency, the fluorescent light being thus conducted into the hollow space.
According to a further preferred embodiment of the present invention, further detecting means can be provided in complementary apertures located opposite each of said further detecting means in the hollow space.
These further means can be provided for detecting fluorescent light resulting from different excitation levels of the fluorescent dye to be detected and having different wavelengths consequently.
It follows that this setup permits measurement of a dye at one excitation wavelength, but simultaneously at different wavelengths of the fluorescent light. Since such simultaneous measurements of the fluorescent light at different wavelengths can only be carried out with very great effort by means of the known devices, the present invention permits the time required for carrying out the measurement of the fluorescent light at different wavelengths to be reduced by a factor N in a simple manner, when N stands for the number of different wavelengths of the fluorescent light.
In addition, the further detecting means according to this preferred embodiment can be used for detecting further fluorescent dyes at the same time. It is therefore possible to measure the fluorescent light of different fluorescent dyes with different excitation wavelengths at the same time. Just as in the setup for measuring the fluorescent light at different wavelengths of a fluorescent dye, a reduction of the measurement time by a factor N is obtained also in cases in which several fluorescent dyes are measured, when N stands for the number of fluorescent dyes to be detected at the same time.
According to a further preferred embodiment, a combination of the two above-mentioned measurements is possible. Hence, the fluorescent light of a plurality of fluorescent dyes can be detected in the case of respective different wavelengths.
According to a preferred embodiment, the detecting means are arranged in their respective apertures in the hollow space in a light-tight arrangement. This eliminates a further source of losses for the fluorescent light and stray light.
According to a preferred embodiment, each detecting means can be provided with a photomultiplier means. In addition, each detecting means can be provided with a color filter means which is adapted to the wavelength of the fluorescent light to be detected by said color filter means. If necessary, each detecting means can be provided with a collimator lens.
According to a further preferred embodiment, a blocking filter device can be provided in front of the first aperture of the hollow space, said blocking filter device blocking the light used for excitation and transmitting the fluorescent light to be detected. By means of this measure the laser light used for excitation can be prevented from being scattered into the hollow space where it would have a disadvantageous influence on the detection of the fluorescent light. Such blocking filter devices can be provided in a particularly simple manner, e.g. in the form of absorbing filters (Anlauffiltern) and/or interference filters, especially when monochromatic light is used.
In accordance with an advantageous embodiment, such a blocking filter device can be realized by an interference filter and/or an absorbing filter known in the prior art.